Project Summary/Abstract Chronic comorbid pain or overlapping pain conditions (COPCs) can be devastating from both personal and societal standpoints. Sufferers ? predominantly women ? present with two or more chronic pain disorders. Etiology of COPCs is poorly understood but there is a strong link to emotional stress as a contributing factor. We showed that in female rats with existing neuropathic trigeminal pain (CCI), 3-day forced swimming (FS) stress induces lasting visceral hypersensitivity referred to the lower back. In rats with such comorbid visceral pain, 5-HT3R protein expression is upregulated in the lumbosacral (L6-S1) spinal cord and corresponding dorsal root ganglia (DRG). Consistently, intrathecal blockade of 5-HT3R transiently attenuates referred hyperalgesia. These observations suggest the involvement of 5-HT3R-mediated sensitization of primary afferents from the gut in stress-induced comorbid visceral pain. However, the underlying gut mechanisms remain poorly understood. Accumulating recent evidence supports the importance of gut microbial dysregulation in the pathogenesis of stress-induced visceral pain. But it is unclear whether stress-induced gut dysbiosis mediates cellular and molecular mechanisms underlying the development of visceral pain. We examined fecal microbiota composition of rats with neuropathic trigeminal pain before and after FS stress. Our preliminary data show extensive compositional changes in the gut microbiome of female rats with referred hyperalgesia. Furthermore, fecal microbiota transfer (FMT) from CCI+FS donors induces referred hyperalgesia in CCI recipients with ongoing orofacial pain. This treatment does not much affect nave recipients, which underscores the importance of CCI-induced descending serotonergic facilitation. On the other hand, FMT from nave donors attenuates referred hyperalgesia in CCI+FS recipients. We hypothesize that CCI+FS induces gut dysbiosis that contributes to dysregulation of 5-HT3R in the primary afferents from the gut. This dysregulation is evident only at the protein level implicating a posttranscriptional mechanism, particularly microRNAs. Recent literature supports the role of gut microbiota in regulating host microRNA expression. Therefore, we further hypothesize that in CCI+FS rats, gut dysbiosis leads to reduced expression of 5-HT3R-targeting microRNAs in primary afferents from the gut. This results in increased 5-HT3R protein expression without affecting its mRNA levels. We will use multidisciplinary approaches to test our hypotheses as described in three aims. In aim 1, we will examine the role of 5-HT3R in the pathogenesis of persistent referred low back pain. In aim 2, we will address the role of gut microbial dysbiosis in the development of referred low back pain. In aim 3, we will investigate gut microbiota-associated posttranscriptional mechanisms of altered 5-HT3R expression. Our study is designed to tease out possible causality between peripheral gut mechanisms and molecular underpinnings of comorbid visceral pain. This award will help the applicant build a successful career in studying complex mechanisms of chronic pain disorders and their ever more complex comorbidities.